Recovery and purification of therapeutic proteins accounts for approximately 50% of the manufacturing cost of biological drugs. The general industrial purification process often includes a number of unit operation steps, like extraction, precipitation, as well as anion- and cation-exchange chromatography. Affinity chromatography is the preferred downstream process step due to its high recovery, yield and specificity, but the current cost and limitations of affinity chromatography is very substantial and in many cases prohibitive for a more general use of this unit operation. For a general description of conventional purification procedures including affinity chromatography see e.g. Jason and Rydén 1998 (Jason, J-C and Rydén, L., Protein Purification Principles, high-Resolution, Methods and Applications, 2nd edition, Wiley & sons Inc. New York, 1998).
Conventional affinity chromatography is in general characterized by having a capturing ligand immobilised to a solid phase matrix. The ligand reversibly binds a target molecule present in a fluid such as liquid culture medium or serum. Target molecules are recovered by dissociating the complex at eluting conditions. Commercially available affinity matrices are in a ready to use format including capturing ligands covalently attached to the matrices. In conventional affinity chromatography the dissociation constant, KD, between the ligand and the target protein is in the range of about 10−5-10−7M. Interactions with dissociation constants exceeding 10−10-10−11M are often impossible to use, as the conditions required to dissociate the complex are then the same as those that will result in denaturation of the target proteins.
The prior art include alternative variations of affinity chromatography purification methods described in the literature (Wilchek, M. and Gorecki, M. (1973), A New Approach for isolation of Biologically Active Compounds by Affinity Chromatography: Isolation of Trypsin).
FEBS Letters. 31, 1, 149-152, describes antibodies immobilized on an insoluble material. The antibodies have affinity for a certain ligand attached to a complex of two or more proteins, and are independent of the chemical, physical and biological properties of the complex itself. The immobilized antibody matrix serves as means for concentrating the complex. The adsorbed complex can then be recovered from the column by elution. The authors use the trypsin enzyme reacted with dinitrophenylated soybean trypsin inhibitor (DNP-STI) to form the complex. The complex is adsorbed to anti DNP-column and eluted under conditions that dissociate the antigen-antibody binding. The affinity column is then ready for the next purification cycle. The target trypsin is obtained by separation of the trypsin enzyme—dinitrophenylated soybean trypsin inhibitor complex into its components in a later step.
This procedure is different from the present invention in that the affinity column is reusable and it is the binding between the immobilized agent and the linker that is dissociated during elution and not the bond between linker and target biomolecule.
Another concept described by Hammarbergh, B. et al., (Proc. Natl. Acad. Sci USA, 86, 4367-4371 (1989)), is a fusion protein affinity approach and its use to express recombinant human insulin-like growth factor II. The procedure relates to a recombinant target protein of interest (X) fused between two different affinity protein tails (A and B). The protein (X) has a protease-sensitive site. A cell lysate containing the recombinant tripartite fusion protein is first passed through an affinity column containing a tail B-specific ligand. A mixture of full-length protein and proteolytic fragments containing the C-terminal fusion protein region can thus be obtained. In a second passage through a tail A-specific affinity column, the degraded proteins flow through while full-length fusion protein is retained. After site-specific cleavage of the tails, the protein of interest (X) is obtained by passing the cleavage mixture through a mixed affinity column for tails A and B and collecting the flow-through. The authors describe a procedure to obtain the target protein by expressing the target protein as an integrated part in between a dual affinity protein construct.
This is different from the present invention as the described affinity procedure requires two different affinity columns and that the immobilized ligand on the column and the dual affinity fusion protein is dissociated to recover the target biomolecule. Following the elution step and a regeneration procedure, the affinity columns are ready for the next affinity purification cycle. The target protein is only part of the fusion protein and is obtained following enzymatic degrading steps.
In a review article by, Ford, C. F., Suominen, I, Glatz, C. E. (1991) Fusion Tails for the Recovery and Purification of Recombinant Proteins. Protein Expression and Purification, 2, 95-107, the authors discuss the applications and advantages of using fusion tail systems to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Fusion tails are useful for enhancing recovery methods for industrial downstream processing. Nevertheless, for the purification of target proteins a site for specific enzymatic cleavage is included, allowing removal of the tail after recovery. The article describes the application of fusion proteins with one binding partner having affinity for the ligand immobilized on a matrix. The procedures include an enzymatic cleavage step to recover the target protein from the fusion tail as required.
This is different from the present invention as the described affinity procedure requires that the fusion protein is dissociated from the ligand immobilized on the column matrix to recover the protein. Following the elution step and a regeneration procedure, the affinity column is ready for the next affinity purification cycle. Also, different from the present invention is that the target protein is part of the fusion protein and is only obtained following an enzymatic processing step.
In Rigaut, G. et al. (1991) (A Generic Protein Purification Method for Protein Complex Characterization and Proteom Exploration. Nature Biotechnology, 17, 1030-1032), is described a generic procedure for purification of protein complexes using tandem affinity purification (TAP) tag. The purification requires one affinity step followed by an enzymatic step cleaving the first affinity tag from the complex and a second affinity purification step to recover the target protein complex from the protease. Overall, the method involves two binding partners in combination both for binding to a ligand immobilized to a column matrix and a protease cleavage step to expose the second binding partner.
This is different from the present invention as the described affinity procedure requires that the fusion protein is dissociated from the ligand immobilized on the column matrix to recover protein. Following the elution step and a regeneration procedure, the affinity column is ready for the next affinity purification cycle. Also, different from the present invention is that the target protein is part of the fusion protein and is obtained following an enzymatic processing step.
EP1529844 describes a method for altering the properties of a recombinant target protein involving co-expression of target protein and the binding partner. The target protein and the binding partner form a complex in the cell. The complex formation result in altered properties such as accumulation, stability and/or integrity, sub-cellular localization, post-translational modifications, purification, and phase partitioning behavior of natural or recombinant target proteins expressed in a host organism. The binding partner may provide an affinity tag that enables co-purification of the complex and the target protein contained therein.
This description is different from the present invention as it describes a co-expression of the binder and the target in order to form a complex in the cell. The disclosed method is for alteration of the target protein properties in general, whereas the present invention describes a dual affinity polypeptide specifically designed to facilitate a dedicated purification process, wherein the dual affinity polypeptides needs to possess specific binding properties.
Linder et al., (Linder, M., Nevanen, T., Söderholm, L., Bengs, O. and Teeri, T., 1998, Biotechnology and Bioengineering, 60(5): 642-647) describes the use of CBD in fusionproteins for use as an affinity tag for purification. Some leakage from the column was observed.
Shpigel, E. et al. (Biotechnol. Appl. Biochem. (2000) 31, 197-203, “Expression, purification and application of Staphylococcal Protein A fused to cellulose-binding domain”), describes an example of purifying IgG using Protein A-CBD dual affinity polypeptide.
They claim that they save expensive coupling procedures by choosing immobilization of the Protein A functionality to a solid phase through the cellulose-binding domain (CBD) of a fusion protein. The fusion protein is immobilized on the column before adding the target.
Due to leakage problems this choice of dual affinity molecule is unsuitable for biopharmaceutical applications.
Sano et al. (U.S. Pat. No. 5,328,985) describes a fusion protein consisting of streptavidin and one or two immunoglobulin G (IgG) binding domains of protein A expressed in Escherichia coli. The strepavidin-protein A (ST-PA) fusion protein has functional biotin and IgG binding sites. Sano further describes complexes of the streptavidin-protein A fusion protein, a monoclonal antibody to bovine serum albumin (BSA) and biotinylated horseradish peroxidise. Sano also describes a method of labelling cell using the ST-PA fusion protein. Cells are incubated with an antibody to the cell surface antigen, Thy-1. The chimeric protein biotinylated marker complex is subsequently added to the cell suspension. This technique was used to deliver biotinylated FITC to the surface of the cells having Thy-1 antigens on their surface.
However, Sano does not describe or suggest using the ST-PA fusion protein as a tool for purification purposes nor does he describe a procedure of single use affinity chromatography column materials, nor recovery of a target protein.
WO 97/19957 describes an invention related to delivering toxins or nucleic acids into specific cell types using ST-PA fusion proteins for the purpose. Similar to Sano et al. (vide supra), an antibody recognise a surface antigen on the cell surface. The ST-PA binds to the antibody and facilitates a linkage to a biotinylated toxin bound to the biotin-binding site. However, it is not described or suggested to use the ST-PA fusion protein as a tool for purification purposes.
WO 01/95857 discloses a method and components for extracting toxic substances from mammalian blood. The method includes preparing an affinity column (extracorporeal device) and a procedure for extracorporeal extraction of toxic material from mammalian body fluids in connection with diagnosis or treatment of a mammalian condition or disease. The extracorporeal affinity column exemplified in the patent is made by coupling biotinylated entities to a matrix containing immobilized avidin. The biotinylated entity includes a part that binds strongly to the toxin in the mammalian blood. The toxic material is removed (i.e. immobilized but not recovered by elution from the column) from the blood following a conventional affinity chromatography procedure. The product from the flow through chromatography procedure is purified blood as the target (toxic materials) stays immobilized on the column after the process.
This is different from the present invention as it describes a procedure that bind the target tightly with high affinity in order to remove target from the product. The purification procedure is also different from the present invention as the product does not bind to the affinity column, but flows through and is collected as depleted from the toxic material (the target). The toxic material is not released or recovered.
WO 97/09068 discloses a method and chemical components that alter the equilibrium dissociation constant between two pairs of bio-molecules. The chemical component is a polymer that can be stimulated to change conformation and thus binding efficiency. The polymer is coupled e.g. to a specific site of the binding partner (the ligand) immobilised to the matrix of the affinity chromatography column. WO 97/09068 does not describe or suggest the use of a dual affinity component for affinity purification, nor recovering of target molecules.
In general, methods that will improve the capturing efficiency and simplify the purification process as well as reduce costs are desirable.